This invention relates to artificial chromosome constructs containing foreign nucleic acid sequences, such as viral nucleic acid sequences, and methods of using these constructs for therapy and for recombinant virus production.
Artificial chromosomes are large, DNA-based vectors that have been used extensively in the construction of DNA libraries for complex genome mapping and analysis. Artificial chromosomes have been derived from yeast (yeast artificial chromosomes: YACs), bacteria (bacterial artificial chromosomes: BACs, and P1-derived artificial chromosomes: PACs), and mammals (mammalian artificial chromosomes: MACs), such as humans (human artificial chromosomes: HACs). These vectors include elements derived from chromosomes that are responsible for replication and maintenance, and are capable of stably maintaining large genomic DNA fragments.
Herpes Simplex virus (HSV) is the prototypic human herpes virus. Despite the fact that HSV is a human pathogen, there has been a great deal of interest in using HSV as a therapeutic agent. The HSV genome has been sequenced, and many HSV mutants have been generated and used specifically in this context. Generation of HSV mutants has been carried out by using drug selection or by co-transfection of cells with plasmid DNA, usually modified by insertion of a marker gene, and intact viral DNA. Mutants are identified by screening for either drug resistance or recombination and expression of the marker gene, or by plaque hybridization. Another method that has been used to generate herpes virus mutants involves the use of cosmid sets that, taken together, contain complete herpes virus genomes. For example, cosmid sets that contain the entire genomes of Pseudorabies virus (PRV), Varicella-Zoster virus (VZV), Herpes Simplex virus (HSV), Cytomegalovirus (CMV), and Epstein-Barr virus (EBV) have been created. In constructing complete viral genomes from these cosmids, viral sequences are released from the cosmid backbones and transfected into cells. Viral plaques are produced via recombination between the overlapping fragments, which together represent the entire genome. Specific mutations are made in the viral genomes by manipulating the cosmid DNA.